Androgens: They Don’t Just M ake a Man Out of You !

1. Androgens: They Don’t Just Make a Man Out of You ! Aritro Sen Ph.D. University of Rochester School of Medicine & Dentistry Department of Medicine Division of Endocrinology & Metabolism

4. Androgens: They Don’t Just Make a Man Out of You !

5. Hyperandrogenism is Detrimental to Women’s Health

6. Androgen Priming may work… The Egg Timer is about to (((Ring)))

7. If Too Many Androgens Cause Unregulated Follicle Overgrowth, Then What Happens When There Are Too Few Androgen?

8. Androgen Receptor Knockout Mice Have Decreased Fertility • Defective folliculogenesis • Subfertility • Premature ovarian Failure Hu et.al., (2004) PNAS 101 ; 11209-11214 Shiinaet.al., (2006) PNAS 103 ; 1 224-229

9. Androgen Receptor is Expressed Throughout the Hypothalamus-Pituitary-Gonadal axis Granulosa cells Hypothalamus Theca cells Pituitary Oocytes Ovary The Follicle Uterus

10. Generation of Granulosacell (GC)-specific Androgen Receptor knockout (ARKO) Mouse Model + X Amh-RII cre - GC AR loxp/loxp (From Dr. Guido Verhoeven) (From Dr. Martin Matzuk)

11. AR mRNA levels are low in the granulosacells (GC) of GC-ARKO mice GC Pituitary Hypothalamus Relative expression of AR mRNA * WT GC AR +/- GC AR -/- Sen et.al., Mol Endo 2010

12. AR Protein levels are low in the granulosacells (GC) of GC-ARKO mice +/+ +/- -/- AR GC Actin Sen et.al., Mol Endo 2010

13. AR is Expressed in Theca but not Granulosacells in GC-ARKO Mice I II III 20X 40X 20X WT * * GC * GC GC * * GC AR -/- * GC 40X 20X Sen et.al., Mol Endo 2010

14. GC-ARKO Mice Have Grossly Normal Reproductive Organs Ovary Uterus GC AR +/- GC AR -/-

15. Fertility Test

16. GC-ARKO Mice Have Less Frequent and Smaller Litters A. 8-9 week old animals B. 24-25 week old animals Sen et.al., Mol Endo 2010

17. Why do GC-ARKO Mice Have Less Frequent Litters ? GC-ARKO Mice Cycle Abnormally P D GC AR +/- E Stages of Estrus Cycle P GC AR -/- D E 0 10 20 30 Days Sen et.al., Mol Endo 2010

18. GC-ARKO Mice Have Decreased Ovulation Why do GC-ARKO Mice Have Smaller Litter Size? Number of oocytes * GC AR +/- * GC AR -/- Number of super- ovulated oocytes * Sen et.al., Mol Endo 2010

19. GC-ARKO Mice Have Premature Loss of Ovarian Reserve (Premature Ovarian Failure) FSH (ng/ml) *

20. Loss of AR in Granulosa Cells Results in: • Less Frequent Ovulation • Decreased number of oocytes ovulated • Premature Ovarian Failure

21. Follicle Growth in the Ovary

22. GC-ARKO Mice Have a Normal Follicle Distribution at 4-5 Weeks GC AR -/- GC AR +/- % of follicles

23. GC-ARKO Mice Have Decreased Follicle Progression and Increased Follicle Atresia with Age AF AF AF AF AF A CL GC AR -/- A GC AR +/- A AF CL A AF A AF * CL * % of follicles * *

24. Knockout of ARs in GCs Results in Slow Follicular Growth Rate GC AR +/+ GC AR -/- * Follicle diameter (μm) Sen et.al., Mol Endo 2010

25. The AR is Required for Follicle Progression from Pre-Antralto Antral Stage

26. A Ovaries from GC-ARKO Mice Have High Rate of Apoptosis -/- +/- Caspase 3 ] Cleaved Caspase 3 Granulosa cells Actin TUNNEL assay-detects DNA fragmentation that results from apoptosis Sen et.al., Mol Endo 2010 Supplemental Fig 1

28. ANDROGENS (Secret) : Strong enough for a man but made for a woman

29. How do Androgens Promote Follicle Growth and Prevent Follicle Atresia? How do Androgens Signal?

30. Androgen Signaling Cellular Response Androgens Non Genomic Kinases AR AR Genomic Gene Expression TF

31. Extra-nuclear Androgen Signaling Membrane bound EGF ligands Androgens EGFR MMP AR Raf P-Y Ras Src PXN (Matrix Metalloproteinase) MEK ERK Sen et.al. JBC 2010; JCI 2012

32. DHT Promotes PaxillinPhophorylation& Phosphoserine-Paxillin Nuclear Localization Media DHT a b Ras PXN P-PXN Androgens PAXILLIN EGFR AR P-Y P-S PXN Src MMP P-Y P-Y P-S ERK PXN PXN Raf MEK Nucleus Sen et.al., JCI 2012

33. Paxillin is Required for DHT-Induced AR Nuclear Localization DHT Media Ras Androgens Nsp siRNA Androgen Receptor EGFR 1a 1b AR P-Y P-S PXN Src MMP P-Y P-Y P-S PXN siRNA ERK PXN PXN AR Raf 2a 2b MEK Nucleus Sen et.al., JCI 2012

34. How do Androgens Promote Follicle Growth and Prevent Follicle Atresia? AR Atresia

35. Androgen-Induces miRNA-125b Expression in Granulosa Cells * TATAAA Relative expression of miRNA125b 5’ 3’ miR-125b GC Primary Culture (WT) IV III II I ARE: 5’-GGTACAnnnTGTTCT-3’ 5’ UTR region of miR-125b consists of a TATA box and four potential AREs Sen et.al., unpublished

38. Androgen-induced expression of miRNA-125b Requires the Extranuclear Pathway EGFR AR Src P-Y Ras PXN GC Primary Culture (WT) * P-Y MMP P-Y P-S P-S ERK PXN PXN AR Relative expression of miRNA125b Raf MEK PXN siRNA DHT EGF Media U0126 miRN-125b Flutamide Nucleus DHT Sen et.al., unpublished

39. Androgen AR • Extranuclear • MAPK • Paxillin Nuclear (+) miRNA-125b expression Expression of miR-125b in GCs require a synergistic action between the nuclear and extranuclear actions of ARs

40. Targets of miR-125b • Bak1: Bcl-2 homologous killer • Bmf: Bcl2 modifying factor • Bax: bcl2 associated X protein • P53: tumor suppressor protein

41. miRNA-125b Suppresses Pro-Apoptotic Protein Expression in GranulosaCells anti-miR125b inhibitor GC Primary Culture (WT) Nsp Relative expression of miRNA125b Bak * Bax Bmf P53 GAPDH DHT Nsp Anti-miR125b inhibitor Sen et.al., unpublished

42. Androgens miRNA-125b expression Pro-apoptotic proteins Decreased Follicular atresia

43. In GC-ARKO mice, miRNA-125b Expression Correlates with Apoptosis A Relative expression of miR-125b * -/- +/- Caspase 3 ] Cleaved Caspase 3 Granulosa cells WT KO Actin B Bak Bax Bmf P53 GAPDH

44. Androgen AR • Extranuclear • MAPK • Paxillin Nuclear (+) miRNA-125b expression Pro-apoptotic proteins Decreased Follicular atresia

45. How do Androgens Promote Follicle Growth and Prevent Follicle Atresia? AR Atresia

46. Follicles Will Not Grow Without FSH “Androgens augment FSH actions to promote follicular growth” But How ?????

47. Androgens Increase Antral Follicle Number In Whole Ovary Organ Culture Sen et.al., unpublished

48. Androgens Increase Growth of Follicles In Vitro * * Follicular diameter (μm) -DHT +DHT DHT (nM) Sen et.al., unpublished

49. Androgens are not essential for FSHR expression WT KO Relative expression of FSHR mRNA FSHR GAPDH Sen et.al., unpublished

50. In Primary Granulosa Cells, Androgens Increase FSH Protein but not mRNA Expression M DHT FSHR GC Primary Culture (WT) GAPDH Relative expression of FSHR mRNA Sen et.al., unpublished